Summary Background Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, ...but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. Methods This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1–30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 106 CAR-transduced T cells per kg (dose 1), 3 × 106 CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov , number NCT01593696. Findings Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 106 CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0–36·3). The most common non-haematological grade 3 adverse events were fever (nine 43% of 21 patients), hypokalaemia (nine 43% of 21 patients), fever and neutropenia (eight 38% of 21 patients), and cytokine release syndrome (three 14%) of 21 patients). Interpretation CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur. Funding National Institutes of Health Intramural funds and St Baldrick's Foundation.
Chimeric antigen receptor (CAR) T cells represent a potent new approach to treat haematological malignancies. Two CAR T-cell therapies, tisagenlecleucel and axicabtagene ciloleucel, have been ...approved in Europe and the USA, as well as several other countries, for the treatment of leukaemia and lymphoma. These approvals marked a major milestone in the field of cell and gene therapies. However, the clinical development and regulatory evaluation of these innovative therapies faced several challenges that are considered important lessons learned for future similar products. Here, we examine the products' non-clinical and clinical data packages to outline the challenges encountered during the regulatory evaluation process in Europe, and to provide an update on their performance after authorisation.
Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of ...targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.
•We have created a new highly active chimeric antigen receptor (CAR) specific for CD22.•The design of new CARs may benefit more from target antigen epitope selection than from optimizing affinity.
Chimeric antigen receptor natural killer (CAR-NK) cells have remarkable cytotoxicity against hematologic malignancies; however, they may also attack normal cells sharing the target antigen. Since ...human leukocyte antigen DR (HLA-DR) is reportedly lost or downregulated in a substantial proportion of hematologic malignancies, presumably a mechanism to escape immune surveillance, we hypothesize that the anti-cancer specificity of CAR-NK cells can be enhanced by activating them against cancer antigens while inhibiting them against HLA-DR. Here, we report the development of an anti-HLA-DR inhibitory CAR (iCAR) that can effectively suppress NK cell activation against HLA-DR-expressing cells. We show that dual CAR-NK cells, which co-express the anti-CD19 or CD33 activating CAR and the anti-HLA-DR iCAR, can preferentially target HLA-DR-negative cells over HLA-DR-positive cells in vitro. We find that the HLA-DR-mediated inhibition is positively correlated with both iCAR and HLA-DR densities. We also find that HLA-DR-expressing surrounding cells do not affect the target selectivity of dual CAR-NK cells. Finally, we confirm that HLA-DR-positive cells are resistant to dual CAR-NK cell-mediated killing in a xenograft mouse model. Our approach holds great promise for enhancing CAR-NK and CAR-T cell specificity against malignancies with HLA-DR loss.
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CAR-engineered NK cells have a remarkable ability to kill cancer cells, but they may also destroy normal cells sharing the target antigen. To enhance their anti-cancer specificity, Xie and colleagues developed an inhibitory receptor, which can serve as an immunological brake to stop CAR-NK cells from attacking normal cells.
Immunogenicity of CAR T cells in cancer therapy Wagner, Dimitrios L; Fritsche, Enrico; Pulsipher, Michael A ...
Nature reviews. Clinical oncology,
06/2021, Letnik:
18, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Patient-derived T cells genetically reprogrammed to express CD19-specific chimeric antigen receptors (CARs) have shown remarkable clinical responses and are commercially available for the treatment ...of patients with certain advanced-stage B cell malignancies. Nonetheless, several trials have revealed pre-existing and/or treatment-induced immune responses to the mouse-derived single-chain variable fragments included in these constructs. These responses might have contributed to both treatment failure and the limited success of redosing strategies observed in some patients. Data from early phase clinical trials suggest that CAR T cells are also associated with immunogenicity-related events in patients with solid tumours. Generally, the clinical implications of anti-CAR immune responses are poorly understood and highly variable between different CAR constructs and malignancies. These observations highlight an urgent need to uncover the mechanisms of immunogenicity in patients receiving CAR T cells and develop validated assays to enable clinical detection. In this Review, we describe the current clinical evidence of anti-CAR immune responses and discuss how new CAR T cell technologies might impact the risk of immunogenicity. We then suggest ways to reduce the risks of anti-CAR immune responses to CAR T cell products that are advancing towards the clinic. Finally, we summarize measures that investigators could consider in order to systematically monitor and better comprehend the possible effects of immunogenicity during trials involving CAR T cells as well as in routine clinical practice.
Several chemotherapeutic agents and novel immunotherapies provide excellent control of systemic and central nervous system (CNS) leukemia but can be highly neurotoxic. The manifestations of subacute ...methotrexate neurotoxicity are diverse and require vigilant management; nonetheless, symptoms are transient in almost all patients. As methotrexate is a crucial drug to prevent CNS relapse, it is important to aim to resume it after full neurologic recovery. Most children tolerate methotrexate rechallenge without significant delays or prophylactic medications. Neurotoxicity is more frequent with newer immunotherapies such as CD19- chimeric antigen receptor T (CAR T) cells and blinatumomab. A uniform grading system for immune effector cell-associated neurotoxicity syndrome (ICANS) and algorithms for management based on severity have been developed. Low-grade ICANS usually resolves within a few days with supportive measures, but severe ICANS requires multispecialty care in the intensive care unit for life-threatening seizures and cerebral edema. Pharmacologic interventions include anticonvulsants for seizure control and glucocorticoids to reduce neuroinflammation. Anticytokine therapies targeted to the pathophysiology of ICANS are in development. By using illustrative patient cases, we discuss the management of neurotoxicity from methotrexate, CAR T cells, and blinatumomab in this review.
Immunotoxins for leukemia Wayne, Alan S.; FitzGerald, David J.; Kreitman, Robert J. ...
Blood,
04/2014, Letnik:
123, Številka:
16
Journal Article
Recenzirano
Odprti dostop
Unconjugated monoclonal antibodies that target hematopoietic differentiation antigens have been developed to treat hematologic malignancies. Although some of these have activity against chronic ...lymphocytic leukemia and hairy cell leukemia, in general, monoclonal antibodies have limited efficacy as single agents in the treatment of leukemia. To increase their potency, the binding domains of monoclonal antibodies can be attached to protein toxins. Such compounds, termed immunotoxins, are delivered to the interior of leukemia cells based on antibody specificity for cell surface target antigens. Recombinant immunotoxins have been shown to be highly cytotoxic to leukemic blasts in vitro, in xenograft model systems, and in early-phase clinical trials in humans. These agents will likely play an increasing role in the treatment of leukemia.
Background
CD22 is a B‐lineage differentiation antigen that has emerged as a leading therapeutic target in acute lymphoblastic leukemia (ALL).
Procedure
Properties of CD22 expression relevant to ...therapeutic targeting were characterized in primary samples obtained from children and young adults with relapsed and chemotherapy refractory B‐precursor (pre‐B) ALL.
Results
CD22 expression was demonstrated in all subjects (n = 163) with detection on at least 90% of blasts in 155 cases. Median antigen site density of surface CD22 was 3,470 sites/cell (range 349–19,653, n = 160). Blasts from patients with known 11q23 (MLL) rearrangement had lower site density (median 1,590 sites/cell, range 349–3,624, n = 20 versus 3,853 sites/cell, range 451–19,653, n = 140; P = <0.0001) and 6 of 21 cases had sub‐populations of blasts lacking CD22 expression (22%–82% CD22 +). CD22 expression was maintained in serial studies of 73 subjects, including those treated with anti‐CD22 targeted therapy. The levels of soluble CD22 in blood and marrow by ELISA were low and not expected to influence the pharmacokinetics of anti‐CD22 directed agents.
Conclusions
These characteristics make CD22 an excellent potential therapeutic target in patients with relapsed and chemotherapy‐refractory ALL, although cases with MLL rearrangement require close study to exclude the presence of a CD22‐negative blast population. Pediatr Blood Cancer Published 2015. This article is a U.S. Government work and is in the public domain in the USA.
Extracellular vesicles (EVs) deliver bioactive macromolecules (i.e. proteins, lipids and nucleic acids) for intercellular communication in multicellular organisms. EVs are secreted by all cell types ...including immune cells. Immune cell-derived EVs modulate diverse aspects of the immune system to either enhance or suppress immune activities. The extensive effects of immune cell-derived EVs have become the focus of great interest for various nano-biomedical applications, ranging from the medical use of nanoplatform-based diagnostic agents to the development of therapeutic interventions as well as vaccine applications, and thus may be ideal for 'immune-theranostic'. Here, we review the latest advances concerning the biological roles of immune cell-derived EVs in innate and acquired immunity. The intercellular communication amongst immune cells through their EVs is highlighted, showing that all immune cell-derived EVs have their unique function(s) in immunity through intricate interaction(s). Natural-killer (NK) cell-derived EVs, for example, contain potent cytotoxic proteins and induce apoptosis to targeted cancer cells. On the other hand, cancer cell-derived EVs bearing NK ligands may evade immune surveillance and responses. Finally, we discuss possible medical uses for the immune cell-derived EVs as a tool for immune-theranostic: as diagnostic biomarkers, for use in therapeutic interventions and for vaccination.
Extracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) ...cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells.
Abbreviations: NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin
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